1. Field of the Invention:
The present invention relates to immunological methods useful for diagnosing human neurocysticercosis, involving testing for the presence of antigens or antibodies in serum or cerebrospinal fluid (CSF) from a patient to be diagnosed.
2. Discussion of the Background:
In many areas of Central and South America, as well as in areas of Africa, Asia and the South Pacific, it is estimated that 3% to 10% of the people in endemic areas are infected with the larvae of Taenia solium, a condition referred to as cysticercosis. These larvae are acquired by people who accidentally ingest the eggs of the adult tapeworm. The larvae hatch in the intestine and migrate through the body, becoming embedded in various tissues and, particularly, in the brain. If the larvae invade the central nervous system (neurocysticercosis), several neurological problems can develop (i.e., migraine headaches, convulsions, loss of memory, numbness of the limbs, blindness, cerebral hypertension). It is not uncommon to find patients with 15 or more larvae in their brains. These larvae are usually about 1 cm. in diameter but can grow to be larger than 5 cm. in diameter.
It has recently been demonstrated that neurocysticercosis, if diagnosed early, can be readily and effectively treated with Praziquantel, a relatively inexpensive anti-helminthic drug. This drug should not be used unless a positive diagnosis exists. Thus, a reliable diagnosis of neurocysticercosis would be highly desirable for a physician faced with a patient who presents symptoms which may have been brought on by neurocysticercosis. Ultimately, a completely reliable diagnosis of neurocysticercosis requires confirmation of the presence of the larvae in the patient. Current methods of diagnosis are expensive and relatively inaccurate. Physicians in endemic areas who suspect neurocysticercosis on the basis of signs and symptoms depend heavily on CAT scans, if CAT scans are available and if the patient can afford this expensive procedure. In most cases, however, rural clinics and hospitals do not have the radiologic equipment for CAT scans and, at $200 to $300 per CAT scan, most patients cannot afford the procedure. Moreover, it should be emphasized as well that CAT scans cannot discriminate between lesions caused by larvae and other lesions, for example tumors.
Several other methods exist for diagnosis of neurocysticercosis, but each of these methods suffers from some drawback. Laboratory analysis of cerebrospinal fluid (CSF) in neurocysticercosis patients which might lead to diagnosis of this disease has been limited to cytochemical analysis and indirect hemagglutination (IHA). However, cytochemical findings are not specific for neurocysticercosis. Also, positive antibody titers by IHA vary greatly with the clinical presentation of the disease and thus this method is not very reliable.
Recently, enzyme-linked immunosorbent assays (ELISA) have been developed. Mohammad et al. (J. Clin. Microbiol. 20, 775-779, 1984) in a recent study using ELISA for detection of antibodies in serum and CSF of neurocysticercotic patients showed a relative high specificity and sensitivity with this assay (up to 94.7% in CSF from 19 patients). The same authors pointed out that results from ELISA on both CSF and serum in the diagnosis of neurocysticercosis could enhance the reliability of this test. Detection of antibody in serum or cerebrospinal fluid may not indicate that larvae are in the central nervous system since antibody could be in these fluids if larvae are in other parts of the body.
Diwan et al. (Am. J. Trop. Med. Hyg. 31, 364-369, 1982) have described an ELISA procedure for the detection of antibodies in the sera of patients with neurocysticercosis which is 79% accurate. Biagi et al. (Revista Medicina Hospital General 25, 501-507, 1961) and Gutierrez-Moctezuma (Referata Med. ISSSTE (Mexico), 1, 30-34, 1976) have described indirect immunofluorescent assays (IFA) which are 10%-90% accurate. These tests are disadvantageous because (1) they are technically difficult to perform; (2) they detect only antibody and, therefore, may not indicate an active, current infection, and (3) they do not have high sensitivity.
Other studies have been directed to the characterization of antigens in patients suffering from neurocysticercosis which might be useful in diagnosis of the disease. In Mexico, Velasco et al. (Sal. Publ. Mex. 25, 205-208, 1983) found circulating T. solium larval antigens in 77% of 250 CSF samples of neurocysticercosis patients using a latex agglutination test. In the same study, all of the 31 CSF samples from patients in whom cysticercosis was confirmed by surgical exploration were positive for circulating antigens. Following treatment with Praziquantel, additional patients in the same study group were found to have larval antigens in their CSF. This observation suggests that cysticerci within the brain release antigens when patients undergo chemotherapy with Praziquantel. It is important to note that Velasco et al did not characterize the antigenic material at all and referred generally to plural soluble antigens.
Tellez-Giron et al, Am. J. Trop. Med. Hyg. 37, 169-173 (1987) discloses the detection of Cysticercus cellulosae antigens in CSF by dot enzyme-linked immunosorbent assay (dot-ELISA) and a standard assay.
Estrada and Kuhn, J. Neurol. Sci. 71, 39-48 (1985) reported on immunochemical detection of antigens of larval Taenia solium and anti-larval antibodies in the CSF of patients with neurocysticercosis.
The present invention is the first dealing with the characterization of these antigens and the antigens of the vesicular fluid of the larvae infecting the human central nervous system. The characterization and isolation of these antigens can facilitate the production of more specific antibodies and the development of methods of protection against the disease. Furthermore, even though circulating antigens were not demonstrated in all patients, antigen detection methods still offer a more definitive diagnosis than antibody detection which may be nonspecific. Although the immunoblotting technique may not be suitable for routine diagnosis, the information gained from this study can be useful in the development of routine antigen detection methods.
Flisser et al. (Clin. and Exp. Immunol. 39, 27-37, 1980), using immunoelectrophoresis, have studied the types of antibodies produced in the sera of individuals affected with neurocysticercosis, using as antigen, a crude extract of cysticerci. They found a total of eight precipitin bands with one, referred to as antigen B, being the most frequently recognized by the sera of cysticercotic patients. This antigen, which induces the production of IgG antibodies, has been successfully purified. It is composed of two protein subunits, differing in their amino acid compositions, with molecular weights of 95,000 and 105,000 daltons, and an isoelectric point between 5.0 and 5.3 (Guerra et al., Cysticercosis: Present State of Knowledge and Perspectives, Flisser et al (eds.), Academic Press, N.Y., 437-451, 1982). To the best of the inventors' knowledge this is the only antigen of the larval stage of T. solium which has been characterized in detail. It has not been used in a diagnostic technique for neurocysticercosis.
Although there are several diagnostic techniques available for neurocysticercosis, as described above, each of these methods suffers from some degree of unreliability, and there remains a need for new and improved diagnostic techniques based on the detection of particular antigens or antibodies in body fluids of patients which can lead to more reliable diagnosis of neurocysticercosis. The present inventors have found unexpectedly that antibodies in the serum of patients affected with this disease recognize three particular antigens which can be used for an ELISA-based diagnostic technique of neurocysticercosis which is highly reliable. That these particular three larval antigens could serve as the basis for a highly reliable diagnostic test for cysticercosis is totally unexpected in view of the relative nonspecific diagnostic tests of the prior art. Furthermore, it has also been discovered that there are two antigens in cerebrospinal fluid of patients suffering from this disease whose detection in an ELISA technique can serve as the basis for a highly reliable diagnosis of neurocysticercosis also. In consideration of the fact that some antibodies which have been found in the sera of patients may also be found in the sera of persons without the disease, or such antibodies may not be highly correlated with the diagnosis of the disease, it is unpredictable that any test based on the detection of particular antibody or antigen would give as high a reliability as has been observed in the present invention.